Project Summary/Abstract In the last fifteen years, small-angle X-ray scattering (SAXS) has become a key tool in academic, government, and commercial research and development for probing the structure and function of proteins, nucleic acids, and macromolecular complexes. SAXS yields precise but low resolution structural information from biomolecules and complexes in solution, without the need to crystallize or label the biomolecule, regardless of molecular weight or the extent of molecular order. Sophisticated and easy-to-use data analysis suites enable rapid interpretation of SAXS profiles, yielding information ranging from molecular radius of gyration to structural envelopes and ensembles. Most synchrotron sources have dedicated bioSAXS beam lines that allow measurement of hundreds of samples per day, but ongoing efforts to improve their throughput have not kept pace with the rapid expansion in user demand. Currently, all bioSAXS is performed on samples at or near room temperature. Due to biomolecular aggregation and degradation between initial expression/purification and SAXS measurements and to radiation damage by the illuminating X-rays, large volumes of sample are required per measurement. Sample cells must be loaded and then thoroughly cleaned between each measurement, so data collection duty cycles are very low. Large sample volume requirements, low measurement duty cycles, and high user demand are critical bottlenecks in the continued expansion of bioSAXS, especially for high-throughput parameter and ligand interaction screening and for study of difficult to produce proteins or complexes, applications in which bioSAXS may have the greatest impact on human health. Building upon our recent demonstration experiments, this project aims to develop technology and methods for high-throughput SAXS on biomolecular samples at cryogenic temperatures. As with cryocrystallography, cryoSAXS should require much smaller sample volumes per measurement, allow sample preparation in the home lab immediately after purification, easy sample storage and shipping, and automated high-throughput data collection with duty cycles approaching 50%. This will enable dramatically more efficient use of both biomolecules and synchrotron beam time, and significantly expand the potential scope of bioSAXS studies. Key aspects of this technology to be developed are (1) fixed and reproducible path length sample cells and sample cell arrays that enable rapid, homogeneous sample cooling; (2) tools for sample cooling, storage, shipment, and high-throughput handling based on those for high-throughput cryocrystallography; (3) screens of contrast- maximizing cryoprotective buffers of known cryogenic electron density that minimize thermomechanical stresses; (4) measurement-based modeling of hydration layers at cryogenic temperatures, needed for interpretation of cryoSAXS profiles; and (5) extensions of this technology to SAXS studies on biomolecules in supercooled liquid buffers at temperatures between ~200 and 270 K.